Towards the elucidation of the mechanism of action of small molecule upregulators of utrophin using chemical proteomics
<p>Duchenne Muscular Dystrophy (DMD) is an X-linked recessive and progressive muscle-wasting disease caused by a lack of the cytoskeletal protein dystrophin. There is currently no disease modifying treatment for all patients, although various promising approaches (e.g. exon skipping, read thro...
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| その他の著者: | |
| フォーマット: | 学位論文 |
| 言語: | English |
| 出版事項: |
2017
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| _version_ | 1826316080297017344 |
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| author | Vuorinen, A |
| author2 | Russell, A |
| author_facet | Russell, A Vuorinen, A |
| author_sort | Vuorinen, A |
| collection | OXFORD |
| description | <p>Duchenne Muscular Dystrophy (DMD) is an X-linked recessive and progressive muscle-wasting disease caused by a lack of the cytoskeletal protein dystrophin. There is currently no disease modifying treatment for all patients, although various promising approaches (e.g. exon skipping, read through of stop codons and gene therapy) are being developed and are starting to be approved for clinical use. We aim to develop an oral small molecule upregulator that compensates for the missing dystrophin by replacing it with its autosomal paralogue utrophin. This therapy will be applicable to all patients regardless of their dystrophin mutation and will target skeletal muscle, heart and diaphragm. In partnership with Summit Therapeutics, ezutromid (SMT C1100), a small molecule utrophin modulator that reduces dystrophic symptoms in the mdx mouse, is in a Phase 2 clinical trial.</p>
<p>Ezutromid demonstrates a proof of principle for the strategy, but we still need to rapidly progress follow-on compounds to maximise the success of the utrophin modulation approach. Novel utrophin modulator chemotypes, which showed better activity compared with ezutromid, were discovered using an improved utrophin luciferase knock-in (LUmdx; mdx,utrluc/+) screening cell line. However, the precise mechanism by which these small molecules increase levels of utrophin is not understood. Importantly, the initial evidence shows that some of these small molecules modulate utrophin transcription through an alternative regulatory mechanism to ezutromid.</p>
<p>The aim of this thesis was to identify the target(s) and elucidate the mechanism of action using one of these new utrophin modulators, OX01914, as a lead compound. Firstly, structure activity relationship studies (SAR) were conducted to find a suitable linker type and position within OX01914 scaffold. As a result, biotinylated pull-down probes and a corresponding inactive analog were synthesised. However, the probes, which were predicted to be active based on previous SAR studies, did not show any activity in the LUmdx reporter assay. This was envisaged to be attributable to poor cell permeability of biotin tagged derivatives and supported by the activity of corresponding acetamido substituted probe.</p>
<p>To overcome the poor cell permeability of the biotin tag, clickable probes with an alkyne tag were synthesised. In addition, to circumvent the possible weak binding affinity between the probe and the target(s), dual tagged probes bearing a photoaffinity label (diazirine) were synthesised. These probes retained sufficient activity compared with OX01914 and the initial Cu(I)-catalysed click chemistry reactions were carried out. However, it was discovered that copper is incompatible with the acyl hydrazine head group of OX01914. Therefore, strain promoted azide-alkyne cycloaddition (SPAAC) was proposed as an alternative strategy and the synthesis of azide tagged probes and a corresponding dual tagged probe was carried out. These probes exhibit sufficient activity. In addition, the results from preliminary photolysis and SPAAC experiments suggested that these probes could be used in future in situ pull-down experiments.</p>
<p>BODIPY FL tagged probes were synthesised and used for preliminary cellular imaging. Finally, pull-down experiments were carried out using LUmdx cell lysate, active biotinylated probe, its inactive analog and corresponding free competition controls. As a result, several significant proteins were identified by active biotinylated probe. Two of these proteins, annexin A1 and β-catenin, were chosen for subsequent target validation studies due to their relevance to DMD. Although, Western blotting results using annexin A1 and β-catenin antibodies were inconclusive, WaterLOGSY experiments using recombinant annexin A1 supported the mass spectrometry data supporting annexin A1 as a possible molecular target for these compounds.</p> |
| first_indexed | 2024-03-07T00:53:31Z |
| format | Thesis |
| id | oxford-uuid:8734b8c8-c48f-45bc-a5ee-0ecd6b27b978 |
| institution | University of Oxford |
| language | English |
| last_indexed | 2024-12-09T03:39:30Z |
| publishDate | 2017 |
| record_format | dspace |
| spelling | oxford-uuid:8734b8c8-c48f-45bc-a5ee-0ecd6b27b9782024-12-05T14:53:00ZTowards the elucidation of the mechanism of action of small molecule upregulators of utrophin using chemical proteomicsThesishttp://purl.org/coar/resource_type/c_db06uuid:8734b8c8-c48f-45bc-a5ee-0ecd6b27b978EnglishORA Deposit2017Vuorinen, ARussell, A<p>Duchenne Muscular Dystrophy (DMD) is an X-linked recessive and progressive muscle-wasting disease caused by a lack of the cytoskeletal protein dystrophin. There is currently no disease modifying treatment for all patients, although various promising approaches (e.g. exon skipping, read through of stop codons and gene therapy) are being developed and are starting to be approved for clinical use. We aim to develop an oral small molecule upregulator that compensates for the missing dystrophin by replacing it with its autosomal paralogue utrophin. This therapy will be applicable to all patients regardless of their dystrophin mutation and will target skeletal muscle, heart and diaphragm. In partnership with Summit Therapeutics, ezutromid (SMT C1100), a small molecule utrophin modulator that reduces dystrophic symptoms in the mdx mouse, is in a Phase 2 clinical trial.</p> <p>Ezutromid demonstrates a proof of principle for the strategy, but we still need to rapidly progress follow-on compounds to maximise the success of the utrophin modulation approach. Novel utrophin modulator chemotypes, which showed better activity compared with ezutromid, were discovered using an improved utrophin luciferase knock-in (LUmdx; mdx,utrluc/+) screening cell line. However, the precise mechanism by which these small molecules increase levels of utrophin is not understood. Importantly, the initial evidence shows that some of these small molecules modulate utrophin transcription through an alternative regulatory mechanism to ezutromid.</p> <p>The aim of this thesis was to identify the target(s) and elucidate the mechanism of action using one of these new utrophin modulators, OX01914, as a lead compound. Firstly, structure activity relationship studies (SAR) were conducted to find a suitable linker type and position within OX01914 scaffold. As a result, biotinylated pull-down probes and a corresponding inactive analog were synthesised. However, the probes, which were predicted to be active based on previous SAR studies, did not show any activity in the LUmdx reporter assay. This was envisaged to be attributable to poor cell permeability of biotin tagged derivatives and supported by the activity of corresponding acetamido substituted probe.</p> <p>To overcome the poor cell permeability of the biotin tag, clickable probes with an alkyne tag were synthesised. In addition, to circumvent the possible weak binding affinity between the probe and the target(s), dual tagged probes bearing a photoaffinity label (diazirine) were synthesised. These probes retained sufficient activity compared with OX01914 and the initial Cu(I)-catalysed click chemistry reactions were carried out. However, it was discovered that copper is incompatible with the acyl hydrazine head group of OX01914. Therefore, strain promoted azide-alkyne cycloaddition (SPAAC) was proposed as an alternative strategy and the synthesis of azide tagged probes and a corresponding dual tagged probe was carried out. These probes exhibit sufficient activity. In addition, the results from preliminary photolysis and SPAAC experiments suggested that these probes could be used in future in situ pull-down experiments.</p> <p>BODIPY FL tagged probes were synthesised and used for preliminary cellular imaging. Finally, pull-down experiments were carried out using LUmdx cell lysate, active biotinylated probe, its inactive analog and corresponding free competition controls. As a result, several significant proteins were identified by active biotinylated probe. Two of these proteins, annexin A1 and β-catenin, were chosen for subsequent target validation studies due to their relevance to DMD. Although, Western blotting results using annexin A1 and β-catenin antibodies were inconclusive, WaterLOGSY experiments using recombinant annexin A1 supported the mass spectrometry data supporting annexin A1 as a possible molecular target for these compounds.</p> |
| spellingShingle | Vuorinen, A Towards the elucidation of the mechanism of action of small molecule upregulators of utrophin using chemical proteomics |
| title | Towards the elucidation of the mechanism of action of small molecule upregulators of utrophin using chemical proteomics |
| title_full | Towards the elucidation of the mechanism of action of small molecule upregulators of utrophin using chemical proteomics |
| title_fullStr | Towards the elucidation of the mechanism of action of small molecule upregulators of utrophin using chemical proteomics |
| title_full_unstemmed | Towards the elucidation of the mechanism of action of small molecule upregulators of utrophin using chemical proteomics |
| title_short | Towards the elucidation of the mechanism of action of small molecule upregulators of utrophin using chemical proteomics |
| title_sort | towards the elucidation of the mechanism of action of small molecule upregulators of utrophin using chemical proteomics |
| work_keys_str_mv | AT vuorinena towardstheelucidationofthemechanismofactionofsmallmoleculeupregulatorsofutrophinusingchemicalproteomics |